SOX2 Modulates the Nuclear Organization and Transcriptional Activity of the Glucocorticoid Receptor.

Steroid receptors (SRs) are ligand-dependent transcription factors (TFs) relevant to key cellular processes in both physiology and pathology, including some types of cancer. SOX2 is a master TF of pluripotency and self-renewal of embryonic stem cells, and its dysregulation is also associated with various types of human cancers. A potential crosstalk between these TFs could be relevant in malignant cells yet, to the best of our knowledge, no formal study has been performed thus far. Here we show, by quantitative live-cell imaging microscopy, that ectopic expression of SOX2 disrupts the formation of hormone-dependent intranuclear condensates of many steroid receptors (SRs), including those formed by the glucocorticoid receptor (GR). SOX2 also reduces GR's binding to specific DNA targets and modulates its transcriptional activity. SOX2-driven effects on GR condensates do not require the intrinsically disordered N-terminal domain of the receptor and, surprisingly, neither relies on GR/SOX2 interactions. SOX2 also alters the intranuclear dynamics and compartmentalization of the SR coactivator NCoA-2 and impairs GR/NCoA-2 interactions. These results suggest an indirect mechanism underlying SOX2-driven effects on SRs involving this coactivator. Together, these results highlight that the transcriptional program elicited by GR relies on its nuclear organization and is intimately linked to the distribution of other GR partners, such as the NCoA-2 coactivator. Abnormal expression of SOX2, commonly observed in many tumors, may alter the biological action of GR and, probably, other SRs as well. Understanding this crosstalk may help to improve steroid hormone-based therapies in cancers with elevated SOX2 expression.

Power-law behavior of transcription factor dynamics at the single-molecule level implies a continuum affinity model.

Single-molecule tracking (SMT) allows the study of transcription factor (TF) dynamics in the nucleus, giving important information regarding the diffusion and binding behavior of these proteins in the nuclear environment. Dwell time distributions obtained by SMT for most TFs appear to follow bi-exponential behavior. This has been ascribed to two discrete populations of TFs-one non-specifically bound to chromatin and another specifically bound to target sites, as implied by decades of biochemical studies. However, emerging studies suggest alternate models for dwell-time distributions, indicating the existence of more than two populations of TFs (multi-exponential distribution), or even the absence of discrete states altogether (power-law distribution). Here, we present an analytical pipeline to evaluate which model best explains SMT data. We find that a broad spectrum of TFs (including glucocorticoid receptor, oestrogen receptor, FOXA1, CTCF) follow a power-law distribution of dwell-times, blurring the temporal line between non-specific and specific binding, suggesting that productive binding may involve longer binding events than previously believed. From these observations, we propose a continuum of affinities model to explain TF dynamics, that is consistent with complex interactions of TFs with multiple nuclear domains as well as binding and searching on the chromatin template.

Phasing the intranuclear organization of steroid hormone receptors.

Steroid receptors (SRs) encompass a family of transcription factors that regulate the expression of thousands of genes upon binding to steroid hormones and include the glucocorticoid, androgen, progesterone, estrogen and mineralocorticoid receptors. SRs control key physiological and pathological processes, thus becoming relevant drug targets. As with many other nuclear proteins, hormone-activated SRs concentrate in multiple discrete foci within the cell nucleus. Even though these foci were first observed ∼25 years ago, their exact structure and function remained elusive. In the last years, new imaging methodologies and theoretical frameworks improved our understanding of the intranuclear organization. These studies led to a new paradigm stating that many membraneless nuclear compartments, including transcription-related foci, form through a liquid-liquid phase separation process. These exciting ideas impacted the SR field by raising the hypothesis of SR foci as liquid condensates involved in transcriptional regulation. In this work, we review the current knowledge about SR foci formation under the light of the condensate model, analyzing how these structures may impact SR function. These new ideas, combined with state-of-the-art techniques, may shed light on the biophysical mechanisms governing the formation of SR foci and the biological function of these structures in normal physiology and disease.

Unraveling the molecular interactions involved in phase separation of glucocorticoid receptor.

BACKGROUND: Functional compartmentalization has emerged as an important factor modulating the kinetics and specificity of biochemical reactions in the nucleus, including those involved in transcriptional regulation. The glucocorticoid receptor (GR) is a ligand-activated transcription factor that translocates to the nucleus upon hormone stimulation and distributes between the nucleoplasm and membraneless compartments named nuclear foci. While a liquid-liquid phase separation process has been recently proposed to drive the formation of many nuclear compartments, the mechanisms governing the heterogeneous organization of GR in the nucleus and the functional relevance of foci formation remain elusive. RESULTS: We dissected some of the molecular interactions involved in the formation of GR condensates and analyzed the GR structural determinants relevant to this process. We show that GR foci present properties consistent with those expected for biomolecular condensates formed by a liquid-liquid phase separation process in living human cells. Their formation requires an initial interaction of GR with certain chromatin regions at specific locations within the nucleus. Surprisingly, the intrinsically disordered region of GR is not essential for condensate formation, in contrast to many nuclear proteins that require disordered regions to phase separate, while the ligand-binding domain seems essential for that process. We finally show that GR condensates include Mediator, a protein complex involved in transcription regulation. CONCLUSIONS: We show that GR foci have properties of liquid condensates and propose that active GR molecules interact with chromatin and recruit multivalent cofactors whose interactions with additional molecules lead to the formation of a focus. The biological relevance of the interactions occurring in GR condensates supports their involvement in transcription regulation.

Assaying Homodimers of NF-κB in Live Single Cells.

NF-κB is a family of heterodimers and homodimers which are generated from subunits encoded by five genes. The predominant classical dimer RelA:p50 is presumed to operate as "NF-κB" in many contexts. However, there are several other dimer species which exist and may even be more functionally relevant in specific cell types. Accurate characterization of stimulus-specific and tissue-specific dimer repertoires is fundamentally important for understanding the downstream gene regulation by NF-κB proteins. In vitro assays such as immunoprecipitation have been widely used to analyze subunit composition, but these methods do not provide information about dimerization status within the natural intracellular environment of intact live cells. Here we apply a live single cell microscopy technique termed Number and Brightness to examine dimers translocating to the nucleus in fibroblasts after pro-inflammatory stimulation. This quantitative assay suggests that RelA:RelA homodimers are more prevalent than might be expected. We also found that the relative proportion of RelA:RelA homodimers can be perturbed by small molecule inhibitors known to disrupt the NF-κB pathway. Our findings show that Number and Brightness is a useful method for investigating NF-κB dimer species in live cells. This approach may help identify the relevant targets in pathophysiological contexts where the dimer specificity of NF-κB intervention is desired.

The glucocorticoid receptor interferes with progesterone receptor-dependent genomic regulation in breast cancer cells.

The glucocorticoid and progesterone receptors (GR and PR) are closely related members of the steroid receptor family. Despite sharing similar structural and functional characteristics; the cognate hormones display very distinct physiological responses. In mammary epithelial cells, PR activation is associated with the incidence and progression of breast cancer, whereas the GR is related to growth suppression and differentiation. Despite their pharmacological relevance, only a few studies have compared GR and PR activities in the same system. Using a PR+/GR+ breast cancer cell line, here we report that either glucocorticoid-free or dexamethasone (DEX)-activated GR inhibits progestin-dependent gene expression associated to epithelial-mesenchymal-transition and cell proliferation. When both receptors are activated with their cognate hormones, PR and GR can form part of the same complex according to co-immunoprecipitation, quantitative microscopy and sequential ChIP experiments. Moreover, genome-wide studies in cells treated with either DEX or R5020, revealed the presence of several regions co-bound by both receptors. Surprisingly, GR also binds novel genomic sites in cells treated with R5020 alone. This progestin-induced GR binding was enriched in REL DNA motifs and located close to genes coding for chromatin remodelers. Understanding GR behavior in the context of progestin-dependent breast cancer could provide new targets for tumor therapy.

Glucocorticoid receptor quaternary structure drives chromatin occupancy and transcriptional outcome.

Most transcription factors, including nuclear receptors, are widely modeled as binding regulatory elements as monomers, homodimers, or heterodimers. Recent findings in live cells show that the glucocorticoid receptor NR3C1 (also known as GR) forms tetramers on enhancers, owing to an allosteric alteration induced by DNA binding, and suggest that higher oligomerization states are important for the gene regulatory responses of GR. By using a variant (GRtetra) that mimics this allosteric transition, we performed genome-wide studies using a GR knockout cell line with reintroduced wild-type GR or reintroduced GRtetra. GRtetra acts as a super receptor by binding to response elements not accessible to the wild-type receptor and both induces and represses more genes than GRwt. These results argue that DNA binding induces a structural transition to the tetrameric state, forming a transient higher-order structure that drives both the activating and repressive actions of glucocorticoids.

21-Hydroxy-6,19-epoxyprogesterone: A Promising Therapeutic Agent and a Molecular Tool for Deciphering Glucocorticoid Action.

Glucocorticoids are steroid hormones that exert most of their effects through their binding to the glucocorticoid receptor (GR), a ligand regulated transcription factor. Although glucocorticoids are widely used in the clinic, their usage in chronic therapies provokes severe adverse reactions. In the quest for safer glucocorticoids a dissociated model was established that proposes a disconnection between GR activated pathways responsible of desired pharmacological effects and pathways involved in adverse GR reactions. Under this model, a myriad of steroidal and non-steroidal compounds has been characterized, with most of them still producing side effects. X-ray crystallographic studies followed by molecular dynamics analysis led research to insights on the receptor Ligand Binding Domain (LBD), which undergoes specific ligand dependent conformational changes that influence receptor activities. In this sense, the flexibility of the ligand structure would contribute to the final GR outcome. Here, we review different data of 21-hydroxy-6,19-epoxyprogesterone (21OH-6,19OP), a rigid steroid with potential pharmaceutical interest due to its anti-inflammatory and immunosuppressive activities, lacking several GR adverse reactions. The rigid structure endows this compound with an enhanced selectivity towards GR. Molecular characterization of the GR/21OH-6,19OP complex revealed specific intermediate conformations adopted by the receptor that would explain the influence on GR dimerization and the recruitment of a specific set of GR transcription modulators. We summarize recent data that will contribute to understand the complexity of glucocorticoid response.

Quantifying transcription factor binding dynamics at the single-molecule level in live cells.

Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data. Here, we describe a detailed approach to perform single-molecule tracking (SMT) of transcription factors in living cells to obtain key binding characteristics, namely their residence time and bound fractions. We discuss different types of fluorophores, labeling density, microscope, cameras, data acquisition, and data analysis. Using the glucocorticoid receptor as a model transcription factor, we compared alternate tags (GFP, mEOS, HaloTag, SNAP-tag, CLIP-tag) for potential multicolor applications. We also examine different methods to extract the dissociation rates and compare them with simulated data. Finally, we discuss several challenges that this exciting technique still faces.

Pioneer factors and ATP-dependent chromatin remodeling factors interact dynamically: A new perspective: Multiple transcription factors can effect chromatin pioneer functions through dynamic interactions with ATP-dependent chromatin remodeling factors.

Transcription factor (TF) signaling regulates gene transcription and requires a complex network of proteins. This network includes co-activators, co-repressors, multiple TFs, histone-modifying complexes, and the basal transcription machinery. It has been widely appreciated that pioneer factors, such as FoxA1 and GATA1, play an important role in opening closed chromatin regions, thereby allowing binding of a secondary factor. In this review we will focus on a newly proposed model wherein multiple TFs, such as steroid receptors (SRs), can function in a pioneering role. This model, termed dynamic assisted loading, integrates data from widely divergent methodologies, including genome wide ChIP-Seq, digital genomic footprinting, DHS-Seq, live cell protein dynamics, and biochemical studies of ATP-dependent remodeling complexes, to present a real time view of TF chromatin interactions. Under this view, many TFs can act as initiating factors for chromatin landscape programming. Furthermore, enhancer and promoter states are more accurately described as energy-dependent, non-equilibrium steady states.

DNA binding triggers tetramerization of the glucocorticoid receptor in live cells.

Transcription factors dynamically bind to chromatin and are essential for the regulation of genes. Although a large percentage of these proteins appear to self-associate to form dimers or higher order oligomers, the stoichiometry of DNA-bound transcription factors has been poorly characterized in vivo. The glucocorticoid receptor (GR) is a ligand-regulated transcription factor widely believed to act as a dimer or a monomer. Using a unique set of imaging techniques coupled with a cell line containing an array of DNA binding elements, we show that GR is predominantly a tetramer when bound to its target DNA. We find that DNA binding triggers an interdomain allosteric regulation within the GR, leading to tetramerization. We therefore propose that dynamic changes in GR stoichiometry represent a previously unidentified level of regulation in steroid receptor activation. Quaternary structure analysis of other members of the steroid receptor family (estrogen, androgen, and progesterone receptors) reveals variation in oligomerization states among this family of transcription factors. Because GR's oligomerization state has been implicated in therapy outcome, our findings open new doors to the rational design of novel GR ligands and redefine the quaternary structure of steroid receptors.

Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions.

The estrogen receptor (ER), glucocorticoid receptor (GR), and forkhead box protein 1 (FoxA1) are significant factors in breast cancer progression. FoxA1 has been implicated in establishing ER-binding patterns though its unique ability to serve as a pioneer factor. However, the molecular interplay between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in vivo. Furthermore, the FoxA1 factor is not associated with detectable footprints at its binding sites throughout the genome. These findings support a model wherein interactions between transcription factors and pioneer factors are highly dynamic. Moreover, at a subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1.

Role of 3'-5'-cyclic adenosine monophosphate on the epidermal growth factor dependent survival in mammary epithelial cells.

Epidermal growth factor (EGF) has been suggested to play a key role in the maintenance of epithelial cell survival during lactation. Previously, we demonstrated that EGF dependent activation of PI3K pathway prevents apoptosis in confluent murine HC11 cells cultured under low nutrient conditions. The EGF protective effect is associated with increased levels of the antiapoptotic protein Bcl-XL. Here, we identify the EGF-dependent mechanism involved in cell survival that converges in the regulation of bcl-X expression by activated CREB. EGF induces Bcl-XL expression through activation of a unique bcl-X promoter, the P1; being not only the PI3K/AKT signaling pathway but also the increase in cAMP levels and the concomitant PKA/CREB activation necessary for both bcl-XL upregulation and apoptosis avoidance. Results presented in this work suggest the existence of a novel connection between the EGF receptor and the adenylate cyclase that would have an impact in preventing apoptosis under low nutrient conditions.

Structural Modeling of GR Interactions with the SWI/SNF Chromatin Remodeling Complex and C/EBP.

The glucocorticoid receptor (GR) is a steroid-hormone-activated transcription factor that modulates gene expression. Transcriptional regulation by the GR requires dynamic receptor binding to specific target sites located across the genome. This binding remodels the chromatin structure to allow interaction with other transcription factors. Thus, chromatin remodeling is an essential component of GR-mediated transcriptional regulation, and understanding the interactions between these molecules at the structural level provides insights into the mechanisms of how GR and chromatin remodeling cooperate to regulate gene expression. This study suggests models for the assembly of the SWI/SNF-A (SWItch/Sucrose-NonFermentable) complex and its interaction with the GR. We used the PRISM algorithm (PRotein Interactions by Structural Matching) to predict the three-dimensional complex structures of the target proteins. The structural models indicate that BAF57 and/or BAF250 mediate the interaction between the GR and the SWI/SNF-A complex, corroborating experimental data. They further suggest that a BAF60a/BAF155 and/or BAF60a/BAF170 interaction is critical for association between the core and variant subunits. Further, we model the interaction between GR and CCAAT-enhancer-binding proteins (C/EBPs), since the GR can regulate gene expression indirectly by interacting with other transcription factors like C/EBPs. We observe that GR can bind to bZip domains of the C/EBPα homodimer as both a monomer and dimer of the DNA-binding domain. In silico mutagenesis of the predicted interface residues confirm the importance of these residues in binding. In vivo analysis of the computationally suggested mutations reveals that double mutations of the leucine residues (L317D+L335D) may disrupt the interaction between GR and C/EBPα. Determination of the complex structures of the GR is of fundamental relevance to understanding its interactions and functions, since the function of a protein or a complex is dictated by its structure. In addition, it may help us estimate the effects of mutations on GR interactions and signaling.

Melatonin inhibits glucocorticoid-dependent GR-TIF2 interaction in newborn hamster kidney (BHK) cells.

The antagonism exerted by melatonin on the glucocorticoid response has been well established, being strongly dependent on the cellular context. Previously, we found that melatonin inhibits glucocorticoid receptor (GR) dissociation from the chaperone hetero-complex and nuclear translocation on mouse thymocytes. Here, by performing confocal fluorescence microscopy and the Number and Brightness assay we show that in newborn hamster kidney cells (BHK21) melatonin neither affects GR nuclear translocation nor GR homodimerization. Instead, co-immunoprecipitation studies suggest that physiological concentrations of melatonin impair GR interaction with the transcriptional intermediary factor 2 (TIF2). This melatonin effect was not blocked by the MT(1)/MT(2) receptor antagonist luzindole. Curiously, luzindole behaved as an antiglucocorticoid per se by impairing the glucocorticoid-dependent MMTV-driven gene expression affecting neither GR translocation nor GR-TIF2 interaction.

Exploring the molecular basis of action of the passive antiglucocorticoid 21-hydroxy-6,19-epoxyprogesterone.

21-Hydroxy-6,19-epoxyprogesterone (21OH-6,19OP) is a selective antiglucocorticoid that lacks the bulky substituent at C-11 found in active antagonists of the glucocorticoid receptor (GR). Ligand-free GR ligand-binding domain (LBD) and GR LBD complexed with 21OH-6,19OP or the agonist dexamethasone were simulated during 6 ns using molecular dynamics. Results suggest that the time fluctuation and average position adopted by the H1-H3 loop affect the ability of GR LBD-21OH-6,19OP complex to homodimerize, a necessary step in transcriptome assembly. A nuclear localization and a transactivation experiment showed that, although 21OH-6,19OP activates the translocation of the GR, the nuclear complex is unable to induce the transcription of a reporter driven by a promoter, that requires binding to a GR homodimer to be activated. These findings support the hypothesis that the passive antagonist mode of action of 21OH-6,19OP resides, at least in part, in the incapacity of the GR-21OH-6,19OP complex to dimerize.